Recombinant Rat Fibroblast growth factor 2 (Fgf2), partial (Active)

What is Recombinant Rat Fibroblast growth factor 2 (FGF2)?

Recombinant Rat Fibroblast growth factor 2 (FGF2) is a protein that plays crucial roles in regulating multiple cellular processes including cell survival, division, differentiation, migration, and angiogenesis. It functions as a potent mitogen in vitro and can induce angiogenesis in various experimental models. The recombinant protein is typically produced in E. coli expression systems and encompasses amino acid positions 11-154 of the native rat sequence, with a molecular mass of approximately 16.2 kDa .

What are the structural characteristics of Recombinant Rat FGF2?

Recombinant Rat FGF2 belongs to the heparin-binding growth factors family. The protein sequence of the partial active form (11-154aa) contains critical functional domains responsible for receptor binding and biological activity. It is typically produced as a Tag-Free protein with >95% purity as determined by SDS-PAGE. The recombinant protein maintains the native three-dimensional structure necessary for biological activity, although it represents only a portion of the full sequence (9-154aa in the full-length protein) .

What receptors does Recombinant Rat FGF2 interact with?

Recombinant Rat FGF2 acts as a ligand for multiple fibroblast growth factor receptors including FGFR1, FGFR2, FGFR3, and FGFR4. In addition to FGF receptors, it also functions as an integrin ligand, specifically binding to integrin ITGAV:ITGB3 (αVβ3) heterodimer. This interaction with integrin is required for proper FGF2 signaling, creating a complex signaling network that coordinates cellular responses to FGF2 stimulation .

What different isoforms of Rat FGF2 exist and how do they differ?

Rat FGF2 naturally occurs in three distinct isoforms, all translated from a single mRNA through alternative translation initiation sites. These include one low molecular weight isoform (18 kD) and two high molecular weight isoforms (21 kD and 23 kD). The 18 kD isoform predominantly localizes to the cytoplasm, while the 21 kD and 23 kD variants are primarily found in the nucleus. This differential subcellular localization suggests distinct biological functions for these isoforms .

How are FGF2 isoforms differentially regulated in rat tissues?

The expression and regulation of FGF2 isoforms vary significantly across different rat tissues and physiological conditions. In sensory ganglia and peripheral nerves, the isoforms show differential regulation following nerve injury. Similarly, in the adrenal medulla, the expression patterns of FGF2 isoforms change during post-natal development and in response to hormonal stimuli. These tissue-specific and condition-dependent regulations suggest specialized functions for each isoform in different biological contexts .

What is the significance of post-translational modifications in Rat FGF2?

Post-translational modifications play important regulatory roles in FGF2 function. Notably, phosphorylation at Tyrosine-81 (Tyr-81) has been identified as a key regulatory mechanism for FGF2 unconventional secretion. Unlike proteins that follow the classical secretory pathway, FGF2 lacks a signal peptide and is secreted through an unconventional secretion mechanism that is regulated by this specific phosphorylation event .

How should Recombinant Rat FGF2 be reconstituted for experimental use?

For optimal experimental results, reconstitution should follow these protocols: First, briefly centrifuge the vial containing lyophilized FGF2 to collect the powder at the bottom. Reconstitute the protein in deionized sterile water to achieve a concentration between 0.1-1.0 mg/mL. For long-term storage, add glycerol to a final concentration of 5-50% and prepare small aliquots to avoid repeated freeze-thaw cycles that could compromise protein activity .

What cell types are most responsive to Recombinant Rat FGF2 stimulation?

PC12 cells and Schwann cells show notable responsiveness to FGF2 stimulation, making them excellent model systems for studying FGF2 biology. In particular, immortalized Schwann cells and PC12 cells that stably overexpress different FGF2 isoforms demonstrate dramatic modifications in cell proliferation and survival under both serum-free and serum-containing conditions. These cell types can be used to investigate isoform-specific effects on cellular functions and signaling pathways .

How can researchers measure the biological activity of Recombinant Rat FGF2?

The biological activity of Recombinant Rat FGF2 can be assessed through multiple assays targeting different functional aspects:

• Proliferation assays: Measure cell division rates in responsive cell lines like PC12 or Schwann cells

• Cell survival assays: Quantify protection against apoptosis in serum-starved conditions

• Migration assays: Evaluate cellular motility using wound healing or transwell assays

• Angiogenesis assays: Assess tube formation in endothelial cell cultures or CAM assays

• Receptor phosphorylation assays: Detect activation of downstream signaling molecules like ERK1/2

These functional readouts provide comprehensive assessment of FGF2 activity beyond simple binding assays .

How do endogenous-overexpressed versus exogenous-applied FGF2 isoforms differ in their biological effects?

Research has revealed significant differences between the biological effects of endogenous-overexpressed FGF2 isoforms and exogenously applied recombinant FGF2 proteins. When different FGF2 isoforms are stably overexpressed in PC12 cells and immortalized Schwann cells, they produce dramatically different effects on cell proliferation and survival, particularly when tested under serum-free and serum-containing conditions. In contrast, when recombinant FGF2 isoforms are applied exogenously to normal PC12 and immortalized Schwann cells, they produce similar biological effects on cell proliferation and survival. This disparity suggests that the intracellular location and context of FGF2 expression significantly influence its biological functions .

What signaling pathways are activated by Recombinant Rat FGF2?

Recombinant Rat FGF2 activates multiple downstream signaling cascades with context-dependent outcomes:

FGF2 notably mediates phosphorylation of ERK1/2, which promotes retinal lens fiber differentiation. The specific pathway activation pattern depends on cell type, receptor expression profile, and the presence of co-receptors or modulatory factors .

How does Recombinant Rat FGF2 regulate gene expression in target cells?

FGF2 regulates gene expression through multiple mechanisms. Upon binding to its receptors, it activates signaling cascades that ultimately influence transcription factor activity. In PC12 cells, FGF2 treatment affects the mRNA levels of FGF2 receptors themselves, suggesting a feedback regulatory mechanism. Additionally, it modulates the expression of tyrosine hydroxylase, a key enzyme in catecholamine biosynthesis, in PC12 cells. The high molecular weight isoforms (21/23 kD) that localize to the nucleus may directly influence gene expression through interactions with nuclear proteins, though the exact mechanisms remain to be fully elucidated .

What are the optimal experimental conditions for studying FGF2-induced angiogenesis?

When designing experiments to study FGF2-induced angiogenesis, researchers should consider several critical parameters:

• Concentration range: Typically 5-50 ng/mL for in vitro studies, with dose-response curves recommended

• Endothelial cell selection: Human umbilical vein endothelial cells (HUVECs) or rat brain endothelial cells for species-appropriate responses

• Matrix components: Matrigel or collagen matrices supplemented with heparin sulfate proteoglycans

• Incubation period: 4-24 hours for gene expression changes, 24-72 hours for tubule formation

• Quantification methods: Branch point counting, tubule length measurement, and advanced image analysis

The angiogenic response is significantly enhanced when FGF2 is presented in combination with extracellular matrix proteins that contain heparin-binding domains, as these interactions stabilize FGF2 and promote receptor clustering .

How can researchers differentiate between the effects of different FGF2 isoforms?

To differentiate between the effects of different FGF2 isoforms, researchers can employ several strategic approaches:

• Isoform-specific expression constructs: Generate stable cell lines expressing individual FGF2 isoforms with appropriate subcellular targeting signals

• Mutational analysis: Introduce mutations at alternative translation start sites to selectively express specific isoforms

• Subcellular fractionation: Isolate cytoplasmic versus nuclear fractions to assess isoform-specific activities

• Isoform-selective antibodies: Use antibodies that specifically recognize high or low molecular weight isoforms

• Comparative transcriptomics: Analyze gene expression profiles induced by different isoforms

These approaches can reveal the unique biological roles and regulatory mechanisms associated with each FGF2 isoform .

What controls should be included in experiments using Recombinant Rat FGF2?

Rigorous experimental design requires appropriate controls when working with Recombinant Rat FGF2:

Including these controls helps distinguish specific FGF2-mediated effects from experimental artifacts and enables more precise interpretation of results .

What factors might contribute to reduced activity of Recombinant Rat FGF2 in experimental settings?

Several factors can compromise the activity of Recombinant Rat FGF2 in experimental settings:

• Improper reconstitution: Using buffers with incompatible pH or ionic strength

• Protein degradation: Excessive freeze-thaw cycles or prolonged storage at inappropriate temperatures

• Absence of co-factors: Lack of heparin or heparan sulfate proteoglycans that stabilize FGF2-receptor interactions

• Receptor saturation: Excessive FGF2 concentrations leading to receptor downregulation

• Cell culture conditions: Presence of serum components that may sequester or inactivate FGF2

• Species incompatibility: Using rat FGF2 with cells from distant species where receptor binding affinity may be lower

Researchers should optimize protein handling and experimental conditions to maintain FGF2 bioactivity throughout their studies .

How can researchers resolve contradictory data when studying FGF2 signaling?

When faced with contradictory data in FGF2 research, consider these methodological approaches:

• Context-dependent effects: Examine cell type, culture conditions, and experimental timing as sources of variability

• Isoform-specific responses: Determine which FGF2 isoform is being studied, as the 18 kD versus 21/23 kD isoforms may have opposing effects

• Receptor expression profiling: Quantify FGFR1-4 expression levels, as different receptor compositions can dramatically alter signaling outcomes

• Signaling pathway analysis: Use pathway-specific inhibitors to delineate which downstream cascades are responsible for observed effects

• Extracellular matrix influence: Assess how the presence of different ECM components might modulate FGF2 activity

• Concentration-dependent responses: Establish complete dose-response curves, as FGF2 can exhibit biphasic effects

This systematic approach can help reconcile seemingly contradictory findings by identifying the specific experimental conditions that influence FGF2 activity .

How does Recombinant Rat FGF2 compare to FGF2 from other species?

While FGF2 is highly conserved across mammalian species, important differences exist:

These differences are particularly important when designing cross-species experiments or interpreting results from mixed-species systems. While the core functional domains show high conservation, species-specific differences in post-translational modifications and receptor interactions can influence experimental outcomes .

What emerging research areas are exploring novel applications of Recombinant Rat FGF2?

Cutting-edge research with Recombinant Rat FGF2 is expanding into several promising directions:

• Neural regeneration: Exploring FGF2's potential to promote axonal regrowth and functional recovery after nerve injury

• Stem cell differentiation: Using FGF2 to direct lineage-specific differentiation of rat neural stem cells

• Tissue engineering: Incorporating FGF2 into biomaterial scaffolds for enhanced vascularization and tissue integration

• Cancer biology: Investigating the dual roles of FGF2 in tumor progression versus suppression in rat cancer models

• Exosome-mediated signaling: Examining how FGF2 packaging into exosomes influences paracrine communication

• Drug development: Screening for small molecules that selectively modulate specific FGF2-dependent signaling pathways

These emerging areas highlight the continued relevance of rat FGF2 as both a research tool and potential therapeutic target .